Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: Attenuated IL-2/STAT5 signaling and an enhanced exhaustion phenotype in CD8 + T cells within SMARCA4-deficient tumors (A) Schematic of the workflow for transcriptomic profiling of CD8 + T cells. (B) Volcano plot displaying differentially expressed genes in CD8 + T cells from SMARCA4-KD versus WT tumors. (C–E) Pathway enrichment analyses of genes downregulated in CD8 + T cells from SMARCA4-KD tumors, including Gene Ontology (GO) terms, KEGG pathways, and Reactome pathways. (F) The gene set enrichment analysis (GSEA) plot. (G) Correlation matrix (pie chart) showing the association between IL-2 receptor subunits expression and key T cell exhaustion marker genes in tumor-infiltrating CD8 + T cells. (H) Radar plot comparing the normalized expression levels of genes encoding IL-2 receptor subunits and exhaustion markers in CD8 + T cells. (I) Quantification by flow cytometry of the expression frequencies of PD-1, TIGIT, and TIM-3 on tumor-infiltrating CD8 + T cells. (J and K) Representative multiplex immunofluorescence (mIF) images of SMARCA4-WT and -KD tumor sections stained for PanCK (cyan), CD8 (green), GZMB (white), PD-1 (red), TIGIT (orange), and TIM-3 (yellow). Scale bars, 20 μm. Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance. Statistical significance was determined by unpaired Student’s t test or one-way ANOVA where appropriate.

Article Snippet: Stat5 (D2O6Y) Rabbit mAb , Cell Signaling Technology , Cat#94205; RRID: AB_2737403.

Techniques: Expressing, Marker, Flow Cytometry, Multiplex Assay, Immunofluorescence, Staining

Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: SMARCA4 loss in tumor cells attenuates CD8 + T cell function via NF-κB-mediated suppression of ICAM1 (A) Flow cytometry analysis of surface expression of the exhaustion markers PD-1, TIGIT, and TIM-3 on human CD8 + T cells following co-culture. (B and C) Representative flow cytometry plots showing the production of IFN-γ and TNF-α and the surface expression of IL-2Rα (CD25) by CD8 + T cells following co-culture. (D) Quantification of the frequencies of IFN-γ + , TNF-α + , and IL-2Rα + cells among co-cultured CD8 + T cells. (E) Integrated single-nucleus RNA sequencing (snRNA-seq) analysis comparing IL2-STAT5 signaling activity. y axis: IL2-STAT5 signaling score. (F and G) Incoming and outgoing signaling patterns between major cell types in the TME, as inferred from snRNA-seq. (H) Specific cell-cell communication network illustrating the ICAM signaling pathway from tumor cells to CD8 + T cells in patients with SMARCA4-WT NSCLC. (I) Correlation analysis between SMARCA4 and ICAM1 mRNA expression in TCGA cohorts. (J) Immunohistochemistry staining and quantification of ICAM1 protein expression in tumor tissues from SMARCA4-WT ( n = 10) and -deficient ( n = 10) NSCLC patients. (K) Schematic illustrating the proposed link between SMARCA4 deficiency and impaired NF-κB activation. (L) Immunoblot analysis of ICAM1 and p65 protein levels in SMARCA4-WT H2122 cells treated with the NF-κB inhibitor PTDC or vehicle control. (M) ChIP-qPCR analysis showing NF-κB (p65) binding to a specific site within the ICAM1 promoter in SMARCA4-WT H2122 cells ( n = 3). (N) Dual-luciferase reporter assay in SMARCA4-WT H2122 cells ( n = 3). Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Statistical significance was determined by unpaired Student’s t test or one-way ANOVA where appropriate.

Article Snippet: Stat5 (D2O6Y) Rabbit mAb , Cell Signaling Technology , Cat#94205; RRID: AB_2737403.

Techniques: Cell Function Assay, Flow Cytometry, Expressing, Co-Culture Assay, Cell Culture, RNA Sequencing, Activity Assay, Immunohistochemistry, Staining, Activation Assay, Western Blot, Control, ChIP-qPCR, Binding Assay, Luciferase, Reporter Assay

Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: STAT5 activation mediates the therapeutic effect of the PD-1/IL-2 bsAb in SMARCA4-deficient NSCLC (A) Representative flow cytometry plots showing the expression of the exhaustion markers PD-1, TIGIT, and TIM-3 on activated human CD8 + T cells. (B) Quantification of the mean fluorescence intensity (MFI) of the exhaustion markers PD-1, TIGIT, and TIM-3 on CD8 + T cells from the experiment in (A) ( n = 3). (C) Representative flow cytometry plots showing the production of IFN-γ and TNF-α by CD8 + T cells under the conditions described in (A). (D) Quantification of the frequencies of IFN-γ + and TNF-α + cells among CD8 + T cells ( n = 3). (E) Schematic of the adoptive T cell transfer experiment ( n = 8/group). (F) Representative in vivo bioluminescence images of mice from the indicated groups at different time points. (G) Tumor growth curves, as measured by bioluminescence, for mice in each treatment group. (H) Kaplan-Meier survival curves of mice from the four treatment groups. (I) Quantification of the frequency of donor-derived CD45.2 + CD8 + T cells among total tumor-infiltrating lymphocytes. (J) Flow analysis of donor-derived CD45.2 + CD8 + T cells expressing PD-1, TIGIT, and TIM-3. (K–M) Frequency of IFN-γ + (K–L) and TNF-α + (M) cells among donor-derived CD45.2 + CD8 + T cells. Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance. Statistical significance was determined by unpaired Student’s t test or one-way ANOVA or two-way ANOVA where appropriate.

Article Snippet: Stat5 (D2O6Y) Rabbit mAb , Cell Signaling Technology , Cat#94205; RRID: AB_2737403.

Techniques: Activation Assay, Flow Cytometry, Expressing, Fluorescence, In Vivo, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: PD-1/IL-2 bsAb protects CD8 + T cells from macrophage phagocytosis via STAT5-mediated upregulation of CD47 (A) Schematic of the Cleavage Under Targets and Tagmentation (CUT&Tag) assays workflow. (B) Distribution of STAT5 binding signals relative to transcription start sites (TSS) in CD8 + T cells treated with or without the PD-1/IL-2 bsAb. (C) Genomic annotation of differentially enriched STAT5 binding peaks in the bsAb-treated group. (D) KEGG pathway enrichment analysis of genes associated with STAT5 binding peaks. (E and F) Strategy and Venn diagram for identifying potential STAT5 downstream genes. (G) ChIP-qPCR analysis of STAT5 binding to the promoter regions of selected candidate genes ( n = 3). (H) Genome browser tracks showing STAT5 binding signals at the CD47 locus in control and PD-1/IL-2 bsAb-treated CD8 + T cells. (I) Schematic of the macrophage phagocytosis assay. (J) Representative confocal microscopy images showing macrophages (red) engulfing CD8 + T cells (green). Scale bars, 20 μm. (K) Flow cytometry quantification of the percentage of macrophages that had phagocytosed CD8 + T cells under the indicated conditions ( n = 3). Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance. Statistical significance was determined by unpaired Student’s t test or one-way ANOVA where appropriate.

Article Snippet: Stat5 (D2O6Y) Rabbit mAb , Cell Signaling Technology , Cat#94205; RRID: AB_2737403.

Techniques: Binding Assay, ChIP-qPCR, Control, Phagocytosis Assay, Confocal Microscopy, Flow Cytometry

Journal: Cell Death Discovery

Article Title: Hypoxia promotes progression of cervical cancer by modulating the ATXN3-enhanced P53 stability or STAT5 phosphorylation

doi: 10.1038/s41420-025-02822-0

Figure Lengend Snippet: A IHC detection of HIF1-α, ATXN3, P53, and p-STAT5 proteins in HPV16 + cervical SCC tissues. ( n = 17 HPV16 + SCCs). B Statistical analysis of protein levels in HPV16 + cervical SCC tissues by IHC. ( n = 17 HPV16 + SCCs). C Correlation analysis of protein levels in HPV16 + cervical SCC tissues by IHC. (n = 17 HPV16 + SCCs). D IHC detection of HIF1-α, ATXN3, P53, and p-STAT5 proteins in HPV18 + cervical AC tissues. ( n = 2 HPV18 + ACs). E Statistical analysis of protein levels in HPV18 + cervical AC tissues by IHC. ( n = 2 HPV18 + ACs). F WB assay detecting the protein levels of HIF1-α, ATXN3, P53, and p-STAT5 in HPV16 + cervical SCC tissues and adjacent normal tissues. ( n = 21 HPV16 + SCCs). G Statistical analysis of ATXN3, P53, and p-STAT5 protein levels in HPV16 + cervical SCC tissues and adjacent normal tissues by WB. ( n = 21 HPV16 + SCCs). H Correlation analysis of protein levels in HPV16 + cervical SCC tissues and adjacent normal tissues by WB. ( n = 21 HPV16 + SCCs) I WB assay detecting the protein levels of HIF1-α, ATXN3, P53, and p-STAT5 in HPV18 + cervical AC tissues and adjacent normal tissues. ( n = 4 HPV18 + ACs). J Statistical analysis of ATXN3, P53, and p-STAT5 protein levels in HPV18 + cervical AC tissues and adjacent normal tissues by WB. ( n = 4 HPV18 + ACs). K Correlation analysis of protein levels in HPV18 + AC tissues and adjacent normal tissues by WB. ( n = 4 HPV18 + ACs) The statistical notations used in the figure are:ns P > 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Data are represented as mean ± SD. For comparisons involving the HPV18 + AC group ( n = 2 or 4), the Mann-Whitney U test was used. All other comparisons between two groups were performed using a two-tailed unpaired Student’s t test, while linear regression analysis were performed for panels ( C and H ).

Article Snippet: Primary antibodies targeting HIF-1α (1:1000, 36169S, Cell Signaling Technology), ATXN3 (1:1000, MAB5360, Sigma), p-STAT5 (1:1000, #9314 T, Cell Signaling Technology), STAT5 (1:1000, #25656 T, Cell Signaling Technology), P53 (1:1000, #2527 T, Cell Signaling Technology), and β-actin (1:1000, 66009-1-Ig, Proteintech) were used to specifically bind to the corresponding antigens.

Techniques: MANN-WHITNEY, Two Tailed Test

Journal: Cell Death Discovery

Article Title: Hypoxia promotes progression of cervical cancer by modulating the ATXN3-enhanced P53 stability or STAT5 phosphorylation

doi: 10.1038/s41420-025-02822-0

Figure Lengend Snippet: A Effect of ATXN3 overexpression on P53 and p-STAT5 protein levels in C33A, HeLa, and SiHa cells. (n ≥ 3 independent cell cultures). B Statistical analysis of the effect of ATXN3 overexpression on p-STAT5 protein levels in C33A, HeLa, and SiHa cells. Each individual data point represents an independent cell culture experiment. C Statistical analysis of the effect of ATXN3 overexpression on P53 protein levels in C33A, HeLa, and SiHa cells. Each individual data point represents an independent cell culture experiment. D Effect of ATXN3 knockdown on P53 and p-STAT5 protein levels in C33A, HeLa, and SiHa cells. ( n = 3 independent cell cultures). E Statistical analysis of the effect of ATXN3 knockdown on p-STAT5 protein levels in C33A, HeLa, and SiHa cells. Each individual data point represents an independent cell culture experiment. F Statistical analysis of the effect of ATXN3 knockdown on P53 protein levels in C33A, HeLa, and SiHa cells. Each individual data point represents an independent cell culture experiment. G Changes in ATXN3, P53, and p-STAT5 protein levels in C33A, HeLa, and SiHa cells after ATXN3 overexpression or knockdown under hypoxic conditions. ( n = 3 independent cell cultures). H Statistical analysis of changes in ATXN3, P53, and p-STAT5 protein levels in C33A, HeLa, and SiHa cells after ATXN3 overexpression or knockdown under hypoxic conditions. Each individual data point represents an independent cell culture experiment. The statistical notations used in the figure are:ns P > 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Data are represented as mean ± SD. Two-tailed unpaired Student’s t -test was employed for analyses in panels ( B , C , E and F ), while One-way ANOVA were performed for panels ( H ).

Article Snippet: Primary antibodies targeting HIF-1α (1:1000, 36169S, Cell Signaling Technology), ATXN3 (1:1000, MAB5360, Sigma), p-STAT5 (1:1000, #9314 T, Cell Signaling Technology), STAT5 (1:1000, #25656 T, Cell Signaling Technology), P53 (1:1000, #2527 T, Cell Signaling Technology), and β-actin (1:1000, 66009-1-Ig, Proteintech) were used to specifically bind to the corresponding antigens.

Techniques: Over Expression, Cell Culture, Knockdown, Two Tailed Test

Journal: Pharmacological research

Article Title: Ectopic expression of NKG7 enhances CAR-T function and improves the therapeutic efficacy in liquid and solid tumors.

doi: 10.1016/j.phrs.2024.107506

Figure Lengend Snippet: Fig. 4. NKG7 promotes the proliferation of CAR-T cells by enhancing the IL-2 signaling pathway. (A and B) GSEA of DNA replication and cell cycle between B7H3- CAR-TNKG7 and B7H3-CAR-T cells following B7H3 protein stimulation for 6 h. (C) Volcano plot of T cell proliferation-related genes between B7H3-CAR-TNKG7 and B7H3-CAR-T cells. (D) Heatmaps of significant T cell proliferation-related genes. (E and F) IL-2 expression of CAR-T cells was measured by FCM analysis following target cell stimulation for 6 h. (G) Statistics for (E and F). (H) The proliferation of CAR-T cells was detected by a CFSE assay. (I and J) GSEA of the Jak-STAT and PI3K- Akt pathways between B7H3-CAR-TNKG7 and B7H3-CAR-T cells following B7H3 protein stimulation for 6 h. (K) The IL-2 signaling pathway (p-AKT (Ser473), AKT, p- ERK (Thr202/Tyr204), ERK, p-STAT5 (Thr694) and STAT5) in CAR-T cells were explored by WB assays. ***P < 0.001.

Article Snippet: After blocking with 5 % nonfat milk at room temperature for 1 h, the membranes were incubated with primary antibodies against NKG7 (Cat No. 84835, 1:1000, CST), AKT (Cat No. A11016, 1:1000, ABclonal), p-AKT (Ser473) (Cat No. AP0140, 1:1000, ABclonal), ERK (Cat No. 4695, 1:1000, CST), p-ERK (Thr202/Tyr204) (Cat No. 4370, 1:1000, CST), STAT5 (Cat No. 25656, 1:1000, CST), pSTAT5 (Thr694) (Cat No. 4322, 1:1000, CST) and β-actin (Cat No. 66009–1-Ig, 1:5000, Proteintech) overnight at 4◦C.

Techniques: Expressing, Cell Stimulation, CFSE Assay

Journal: International Journal of Molecular Sciences

Article Title: Different Lipid Regulation in Ovarian Cancer: Inhibition of the Immune System

doi: 10.3390/ijms19010273

Figure Lengend Snippet: Normal expression and phosphorylation of the IL-2 receptor and its downstream signaling components. PBLs of healthy individuals or sorted ascites-derived CD4 + CD25 − T cells (5 × 10 5 cells/well) were stimulated with 20,000 αCD3/28 beads/well for three days. Subsequently, samples were stained for flow cytometry analysis. ( A ) Cells were stained for isotype control, CD25, CD122, and CD132. Plots are representative for eight controls and three patients. ( B ) Control ( n = 3) and ascites-derived T cells ( n = 3) were stained for JAK3, STAT5, phospho-STAT5, phosphor-AKT and phosphor-ERK. One representative example is shown.

Article Snippet: The following antibodies were used in this study: CD4-APC (300514, Beckman Coulter, Brea, CA, USA), CD4-FITC (A07750, Beckman Coulter), CD25-PE (555432, BD Biosciences, San Jose, CA, USA), CD122-PE (IM1978, Beckman Coulter), CD132-APC (338608, BioLegend, San Diego, CA, USA), cyclin D (2936P, Cell Signaling, Danvers, MA, USA), cyclin E (551159, Cell Signaling, Danvers, MA, USA), JAK3 (ab45141, Abcam, Cambridge, UK), STAT5 (9363P, Cell Signaling, Danvers, MA, USA), pSTAT5 (9314S, Cell Signaling, Danvers, MA, USA), pAKT (4075S, Cell Signaling, Danvers, MA, USA), and pERK1/2 (4284S, Cell Signaling).

Techniques: Expressing, Phospho-proteomics, Derivative Assay, Staining, Flow Cytometry, Control